Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

Trafficking of Annexins during Membrane Repair in Human Skeletal Muscle Cells.

Defects in membrane repair contribute to the development of muscular dystrophies, such as Miyoshi muscular dystrophy 1, limb girdle muscular dystrophy (LGMD), type R2 or R12. Deciphering membrane repair dysfunctions in the development of muscular dystrophies requires precise and detailed knowledge of the membrane repair machinery in healthy human skeletal muscle cells. Using correlative light and electron microscopy (CLEM), we studied the trafficking of four members of the annexin (ANX) family, in myotubes damaged by laser ablation. Our data support a model in which ANXA4 and ANXA6 are recruited to the disruption site by propagating as a wave-like motion along the sarcolemma. They may act in membrane resealing by proceeding to sarcolemma remodeling. On the other hand, ANXA1 and A2 exhibit a progressive cytoplasmic recruitment, likely by interacting with intracellular vesicles, in order to form the lipid patch required for membrane resealing. Once the sarcolemma has been resealed, ANXA1 is released from the site of the membrane injury and returns to the cytosol, while ANXA2 remains accumulated close to the wounding site on the cytoplasmic side. On the other side of the repaired sarcolemma are ANXA4 and ANXA6 that face the extracellular milieu, where they are concentrated in a dense structure, the cap subdomain. The proposed model provides a basis for the identification of cellular dysregulations in the membrane repair of dystrophic human muscle cells.

Annexin-A6 in Membrane Repair of Human Skeletal Muscle Cell: A Role in the Cap Subdomain.

Defects in membrane repair contribute to the development of some muscular dystrophies, highlighting the importance to decipher the membrane repair mechanisms in human skeletal muscle. In murine myofibers, the formation of a cap subdomain composed notably by annexins (Anx) is critical for membrane repair. We applied membrane damage by laser ablation to human skeletal muscle cells and assessed the behavior of annexin-A6 (AnxA6) tagged with GFP by correlative light and electron microscopy (CLEM). We show that AnxA6 was recruited to the site of membrane injury within a few seconds after membrane injury. In addition, we show that the deficiency in AnxA6 compromises human sarcolemma repair, demonstrating the crucial role played by AnxA6 in this process. An AnxA6-containing cap-subdomain was formed in damaged human myotubes in about one minute. Through transmission electron microscopy (TEM), we observed that extension of the sarcolemma occurred during membrane resealing, which participated in forming a dense lipid structure in order to plug the hole. By properties of membrane folding and curvature, AnxA6 helped in the formation of this tight structure. The compaction of intracellular membranes-which are used for membrane resealing and engulfed in extensions of the sarcolemma-may also facilitate elimination of the excess of lipid and protein material once cell membrane has been repaired. These data reinforce the role played by AnxA6 and the cap subdomain in membrane repair of skeletal muscle cells.

Imaging Membrane Repair in Single Cells Using Correlative Light and Electron Microscopy.

Many cells possess the ability to repair plasma membrane disruption in physiological conditions. Growing evidence indicates a correlation between membrane repair and many human diseases. For example, a negative correlation is observed in muscle where failure to reseal sarcolemma may contribute to the development of muscular dystrophies. Instead, a positive correlation is observed in cancer cells where membrane repair may be exacerbated during metastasis. Here we describe a protocol that combines laser technology for membrane damage, immunostaining with gold nanoparticles and imaging by fluorescence microscopy and transmission electron microscopy (TEM), which allows the characterization of the molecular machinery involved in membrane repair. Fluorescence microscopy enables to determine the subcellular localization of candidate proteins in damaged cells while TEM offers high-resolution ultrastructural analysis of the µm²-disruption site, which enables to decipher the membrane repair mechanism. Here we focus on the study of human skeletal muscle cells, for obvious clinical interest, but this protocol is also suitable for other cell types. © 2018 by John Wiley & Sons, Inc.

Membrane Repair Assay for Human Skeletal Muscle Cells.

The characterization of the membrane repair machinery in human skeletal muscle has become crucial, since it has been shown that some muscular dystrophies result from a defect of this fundamental physiological process. Deciphering membrane repair mechanism requires the development of methodologies allowing studying the response of skeletal muscle cells to sarcolemma damage and identifying candidate proteins playing a role in the membrane repair machinery. Here, we describe a protocol that is based on the creation of cell membrane disruption by infrared laser irradiation in human myotubes. Membrane disruption and repair are assayed by monitoring the incorporation into myotubes of the membrane probe FM1-43. This methodology has recently enabled us to show that Annexin-A5 is required for membrane repair in human skeletal muscle cells (Carmeille et al., Biochim Biophys Acta 1863:2267-2279, 2016).

Membrane repair of human skeletal muscle cells requires Annexin-A5.

Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin.

Annexin-A5 promotes membrane resealing in human trophoblasts.

Annexin-A5 (AnxA5) is the smallest member of the annexins, a group of soluble proteins that bind to membranes containing negatively-charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. AnxA5 presents unique properties of binding and self-assembling on membrane surfaces, forming highly ordered two-dimensional (2D) arrays. We showed previously that AnxA5 plays a central role in the machinery of cell membrane repair of murine perivascular cells, promoting the resealing of membrane damages via the formation of 2D protein arrays at membrane disrupted sites and preventing the extension of membrane ruptures. As the placenta is one of the richest source of AnxA5 in humans, we investigated whether AnxA5 was involved in membrane repair in this organ. We addressed this question at the level of human trophoblasts, either mononucleated cytotrophoblasts or multinucleated syncytiotrophoblasts, in choriocarcinoma cells and primary trophoblasts. Using established procedure of laser irradiation and fluorescence microscopy, we observed that both human cytotrophoblasts and syncytiotrophoblasts repair efficiently a µm²-size disruption. Compared to wild-type cells, AnxA5-deficient trophoblasts exhibit severe defect of membrane repair. Through specifically binding to the disrupted site as early as a few seconds after membrane wounding, AnxA5 promotes membrane resealing of injured human trophoblasts. In addition, we observed that a large membrane area containing the disrupted site was released in the extracellular milieu. We propose mechanisms ensuring membrane resealing and subsequent lesion removal in human trophoblasts. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Control of motor activity in crayfish by the steroid hormone 20-hydroxyecdysone via motoneuron excitability and sensory-motor integration.

We studied the effects of the molting hormone 20-hydroxyecdysone (20E) on leg sensory-motor networks of the red swamp crayfish, Procambarus clarkii. The hormone was injected in isolated crayfish and network activity was analyzed 3 days after injection using electrophysiology on an in vitro preparation of the leg locomotor network. This 20E treatment deeply reduced motor activity, by affecting both intrinsic motoneuron (MN) properties and sensory-motor integration. Indeed, we noticed a general decrease in motor nerve tonic activities, principally in depressor and promotor nerves. Moreover, intracellular recordings of depressor MNs confirmed a decrease of MN excitability due to a drop in input resistance. In parallel, sensory inputs originating from a proprioceptor, which codes joint movements controlled by these MNs, were also reduced. The shape of excitatory post-synaptic potentials (PSPs) triggered in MNs by sensory activity of this proprioceptor showed a reduction of polysynaptic components, whereas inhibitory PSPs were suppressed, demonstrating that 20E acted also on interneurons relaying sensory to motor inputs. Consequently, 20E injection modified the whole sensory-motor loop, as demonstrated by the alteration of the resistance reflex amplitude. These locomotor network changes induced by 20E were consistent with the decrease of locomotion observed in a behavioral test. In summary, 20E controls locomotion during crayfish premolt by acting on both MN excitability and sensory-motor integration. Among these cooperative effects, the drop of input resistance of MNs seems to be mostly responsible for the reduction of motor activity.